There is a continuing need for improved expression vectors exhibiting high levels of expression. In particular expression vectors for use in mammalian cell lines are of increasing importance both for the industrial production of desired polypeptides and for the development of therapies for genetic disorders.
There are many known examples of characterized structural genes, which together with appropriate control sequences may be inserted into suitable vectors and used to transform host cells. A significant problem with the integration of such a structural gene and control regions into the genome of a mammalian cell is that expression has been shown to be highly dependent upon the position of the inserted sequence in the genome. This results in a wide variation in the expression level and only very rarely in a high expression level. The problem of integration site dependence is solved by the present invention and arises from the discovery of sequences referred to herein as "dominant activator sequences" (or "dominant control regions" (DCRs)) which have the property of conferring a cell-type restricted, integration site independent, copy number dependent expression characteristic of a linked gene system.
The human .beta.-like globin genes are a cluster of five genes in the order of 5'-.epsilon.-.sup.9 .gamma.-.sup.A .gamma.-.delta.-.beta.-3', comprising approximately 60 kb of DNA on the short arm of chromosome 11. The different genes are expressed in a developmentally and tissue-specific manner, i.e. the embryonic .epsilon.-gene is expressed in the yolk sac, the fetal .sup.9 .gamma.- and .sup.A .gamma.-genes, primarily in the fetal liver, and the adult .delta.- and .beta.-genes primarily in bone marrow (for a review, see Maniatis et al., Ann. Rev. Genet., (1981) 14, 145-178). Mutations in this gene family constitute the most widespread family of genetic diseases and a large number of these have been characterized, ranging from simple amino acid changes by point mutations to complete deletions of the locus (for a review, see Collins et al, Prog. Nucl. Acid. Res. Mol. Biol., (1984), 31, 315-462). These often lead to severe clinical problems and early death. Conventional treatment of these diseases (transfusions) are costly, risky and, in many cases, inadequate. The present invention provides a therapy for such disorders for example by gene therapy (Hock et al, Nature, (1986), 320, 275-277).
The DNA sequences which regulate the human .beta.-globin gene are located both 5' and 3' to the translation initiation site (Wright et al., Cell, (1984), 38, 265-273; Charnay et al., Cell, (1984), 38, 251-263). Using murine erythroleukemia cells (MEL) and K562 cells at least four separate regulatory elements required for the appropriate expression of the human .beta.-globin gene have been identified; a positively acting globin specific promoter element and a putatively negative reulatory promoter element and two downstream regulatory sequences (enhancers), one located in the gene and one approximately 800 bp downstream from the gene (Antoniou et al, EMBO J., (1988), 7(2), 377-384). A similar enhancer has also been identified downstream of the chicken .beta.-globin gene using cultured chicken erythroid cells (Hesse et al., PNAS U.S.A., (1986), 83, 4312-4316, Choi et al, Nature, (1986), 323, 731-734). The downstream enhancer has been shown to be a developmental stage specific enhancer using transgenic mice (Kollias et al, Cell, (1986), 46, 89-94 and NAR (1987), 15, 5739-5747; Behringer et al., PNAS U.S.A. (1987), 84, 7056-7060. All of these results therefore indicate that multiple development specific control regions of the .beta.-globin are located immediately 5', inside and 3' to the .beta.-globin gene.
However, when a .beta.-globin gene containing all of these control regions is introduced in transgenic mice, the gene is not expressed at the same level as the mouse .beta.-globin gene and exhibits integration site position effects. This is characterized by a highly variable expression of the transgene that is not correlated with the copy number of the injected gene in the mouse genome. The same phenomenon has been observed in almost all the genes that have been studied in transgenic mice (Palmiter et al, Ann. Rev. Genet., (1986), 20, 465-499). Moreover, the level of expression of each injected gene in the case of .beta.-globin is, at best, an order of magnitude below that of the endogenous mouse gene (Magram et al., Nature, (1985), 315, 338-340; Townes et al., EMBO J., (1985), 4 1715-1723; Kollias et al, Cell, (1986), 46, 89-94). A similar problem is observed when the .beta.-globin or other genes are introduced into cultured cells by transfection or retroviral infection. This poses a big problem when considering gene therapy by gene addition in stem cells. It is also a major problem for the expression of recombinant DNA products from tissue cells. Extensive screening for highly producing clones is necessary to identify cell-lines in which the vector is optimally expressed and selection for vector amplification is generally required to achieve expression levels comparable to those of the naturally occurring genes, such as for example .beta.-globin genes in erythroleukemic cell-lines.
The study of the .beta.-globin system has been assisted by analysis of hemoglobinpathies such as thalassemias (van der Ploeg et al, Nature, (1980), 283, 637-642; Curtin et al, In: Haemoglobin Switching: Fifth Conference on Haemoglobin Switching, Washington Ed. G. Stamatoyannopoulos, Alan R. Liss Inc., New York 1987). The Dutch thalassemia is heterozygous for a large deletion which removes 100 kb upstream of the .beta.-globin gene, but leaves the .beta.-globin gene, including all of the control regions described above intact (Kioussis et al, Nature, (1983), 306, 662-666; Wright et al Cell, (1984), 38, 265-273; Taramelli et al, NAR, (1986), 14 7017-7029). Since the patient is heterozygous and transcribes the normal locus in the same nucleus, it indicates that some control mechanism may be overriding the functioning of the control sequences immediately surrounding the genes in the mutant locus. In the case of the Dutch .gamma..beta.-thalassemia there are two possible explanations the observed effects; either a cis acting positive element has been removed or there has been insertion of a negative-acting element in an inactive chromatic configuration and behaves like a classical position effect (Kioussis et al, Nature, (1983), 306, 662-666).
In a chromosome, the genetic material is packaged into a DNA/protein complex called chromatin which has the effect of limiting the availability of DNA for functional purposes. It has been established that many gene systems (including the .beta.-globin system) possess so-called DNase hypersensitive sites. Such sites represent putative regulatory regions, where the normal chromatin structure is altered to allow interaction with trans-acting regulatory regions.
Regions upstream from the epsilon-globin gene and downstream from the .beta.-globin gene which contain a number of "super" hypersensitive sites have been identified. These sites are more sensitive to DNase I digestion in nuclei than the sites found in and around the individual genes when they are expressed (Tuan et al PNAS U.S.A., (1985), 32, 6384-6388; Groudine et al, PNAS U.S.A., (1983), 80, 7551-7555. In addition, they are erythroid cell specific and they are present when any one of the globin genes is expressed.
Tuan et al describe the broad mapping of four major DNase I hypersensitive sites in the 5' boundary area of the ".beta.-like" globin gene. The authors note that certain sequence features of these sites are also found in many transcriptional enhancers and suggest that the sites might also possess enhancer functions and be recognized by erythroid specific cellular factors.
The present invention arose from the discovery that the complete .beta.-globin locus with intact 5' and 3' boundary regions does not exhibit an integration site position dependence. The regions of the locus responsible for this significant characteristic have been determined and shown to correspond to the DNase I super hypersensitive sites. These dominant activator regions are quire distinct from enhancers, exhibiting properties such as integration site independence not exhibited by the known enhancers. The dominant activator sequence used in conjunction with the known promoter/enhancer elements reconstitute full expression of the natural gene. The connection made as part of the present invention between DNase I super hypersensitive sites and dominant activator regions allows the invention to be extended to gene systems other than the .beta.-globin gene system. It has been shown, for example, that a human .beta.-globin gene can be functionally expressed in transgenic mice in an integration site independent manner. Human T-lymphocytes (T-cells) are produced in bone marrow and mature in the thymus where they develop their immunological characteristic of responding to foreign antigens in the body. T-cells are produced in a highly tissue specific manner. It is recognised that T-cells carry specific cellular markers (Bernard et al "Leukocyte Typing", Ed. Bernard et al, p41, Springer Verlag, Berlin and New York, 1984). One such marker is the E-rosette receptor, known by the designation CD2. The structure of the CD 2 marker and the CD2 genes has been the subject of considerable study (Brown et al, Meth. in Enzym., (1987), 150, 536-547 and Lang, G. et al, EMBO J, (1988), 7(6), 1675-1682).
In the latter paper, published after the priority date of the present invention, the property of a large fragment including the CD2 gene to exhibit copy number dependence is noted and a suggestion is made that such a fragment contains a locus forming sequence analogous to those found in .beta.-globin. DNase super hypersensitive sites have now been identified in the fragment and shown to be dominant activator sequences of the present invention.
Finally, the present invention is applicable to the production of transgenic animals and the techniques for producing such are now widely known. For a review, see Jaenisch, Science, (1988), 240, 1468-1474.
The present invention provides a solution to the problem of integration site dependence of expression making possible the insertion of functionally active gene systems into mammalian genomes both in vitro and in vivo. Specific sequences providing advantageous increases in transcription levels have also been identified.